Many integral membrane proteins have been identified as cleavage substrates for the intramembrane aspartyl protease complex termed ??secretase. These ??secretase substrates include receptors for signaling pathways, adhesion molecules, and other factors that are localized at the cell surface and whose activities are regulated by ??secretase-mediated cleavage. Understanding the molecular details of ??secretase substrate biology is important due to the prominent role that many substrates play in normal cellular physiology and in human disease. For example, the Notch receptor, a well- characterized ??secretase substrate, is essential for proper cell-fate specification and cellular differentiation during the development of animal and human tissues. Inappropriate activation of the Notch pathway in the human immune system causes T-cell leukemia and dysregulated Notch activity has been implicated in other cancers and developmental diseases. Furthermore, processing of the Amyloid Precursor Protein (APP) by ??secretase contributes directly to the production of secreted amyloid-2 peptide in human brain tissue, a crucial event in the pathogenesis of Alzheimer's disease. A key feature of ??secretase substrate cleavage is its tight control by ligand binding, ectodomain removal, and other regulatory processes. A growing body of data indicates that cleavage also depends upon intracellular membrane trafficking of ??secretase substrates and their sorting within specific trafficking compartments. In the case of both Notch and APP, ??secretase-mediated cleavage is associated with endocytosis of the substrate from the cell surface and its entry into endosomes. We have found that proper biogenesis of the early endosome compartment requires the function of an aquaporin channel protein, defining a specialized endosome route for efficient ??secretase-dependent Notch trafficking and signaling. In Specific Aim 1, we propose to characterize further the role of this aquaporin in endosome biogenesis and Notch trafficking. These studies will shed light on the cell biological aspects of ?? secretase substrate trafficking and their relationship to ??secretase substrate cleavage, recycling, and degradation. In Specific Aim 2, we propose to conduct a forward genetic screen for new factors involved in the intracellular membrane trafficking of Notch and other ??secretase substrates. Preliminary results from this screen have already proven to be very promising, including the recovery of many potentially new genes needed for various steps of Notch intracellular trafficking. Further analysis and molecular cloning of several of these new genes is now underway. The main goal of this project is to advance our understanding of ??secretase substrate trafficking and its relevance to secretase- dependent processes in tumorigenesis and neurodegenerative disease. PUBLIC HEALTH RELEVANCE: Proteins cleaved by ??secretase, including the Notch receptor and amyloid precursor protein, are important for developmental signaling and neuronal function, and their dysregulation contributes to cancer and neurodegenerative disease. This research proposal seeks to identify cellular factors that control the production, trafficking, and proteolytic processing of ?? secretase targets. These studies might lead to the identification of new proteins and intracellular pathways that could be therapeutically targeted in cancer and neurodegenerative disorders.